The present invention concerns new cephalotaxanes, their methods of preparation and their use in treatment of cancers, leukemias, parasites including thus resistant to usual chemotherapeutic agents and as reversal agents of harringtonines.
Cephalotaxanes (CTX) are particular alkaloids today only extracted from the Cephalotaxaceae family which exhibiting the structural formula 1. 
Several substituents may be encountered on this core: hydroxyl, ether, acyloxy etc. Some double bound or intramolecular bridge achieve to definite cephalotaxanes. Several dozen of cephalotaxanes have been isolated from various cephalotaxus species.
Cephalotaxoids include cephalotaxanes and unnatural analogs of cephalotaxanes.
Cephalotaxines 2 are cephalotaxanes without acyloxy side-chain.
Harringtonines (i.e. harringtonine=HA and homoharringtonine=HHT) are natural esters of cephalotaxines exhibiting generally a strong cytotoxic activity,
Harringtonines are natural esters of cephalotaxines exhibiting generally a strong cytotoxic activity.
Harringtoids include harringtonines and unnatural analogs of harringtonines.
Two harringtonines are very promising drugs in the treatment of certain leukemia such as Chronic Myelogenous Leukemia (CML). Definite activity of HHT was observed in acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL), and myelodysplastic syndrome (MDS) (Warrell Jr Rpet al, 3:617-621, 1985; Feldman E et al., Leukemia 6:1185-88, 1992; Feldman E J et al, Leukemia 10:40-42, 1996; Kantarjian H et al., Cancer 63:813-817, 1989; Kantarjian H et al., J Clin Oncol 18:3513-3521,2000). The present applicant have initiated in France compassionate use of HHT in CML patients resistant or not eligible to all existing therapies and several phase II and III clinical trials including in patient with CML and AML are ongoing in France and in the U.S. However, it should be pointed out that harringtonines pertain to the series of natural drugs exhibiting the multiresistance phenomenon which led to relapse of the cancer diseases. This situation is a serious limitation to the use of natural chemotherapeutic agents in the treatment of cancers and leukemia.
Harringtonine inhibit protein synthesis at the level of elongation, however ultimate mechanism of action of harringtonine remain unknown. The final result is the self-destruct of the cell. Clinically, harringtonines have a selective action in leukemia of the myeloid series. In addition, harringtonines interacts with P Glycoprotein (PGP) and Multiresistance Protein (MRP). PGP, MRP and other efflux pumps are complex molecular entities which are ubiquist in nature. Their role is to selectively efflux the environmental natural toxic agents, including agents of chemotherapy (anthracyclines, taxanes, epipodophyllotoxins, harringtonine, etc.) It was pointed out that no common structural feature of this natural cytotoxic may related to molecular recognizing by PGP.
A number of analogs all less active than harringtonines have been synthesized. The more active among these esters are about one magnitude less cytotoxic than harringtonines in vitro (i.e. HA, HHT neoharringtonine, have an activity=IC50 ranged from 10 to 30 ng per mL, whereas analog previously synthesized have an IC50 higher than 100 ng/mL). No relation structure activity relationship had been previously found since the discovering of harringtonines.
Therefore, there is the need of new analogs of harringtonines having the same magnitude of cytotoxicity than harringtonines in vitro.
Surprisingly, the present applicant have synthesized a series of CTX analogs exhibiting stronger in vitro inhibition of leukemic cell lines such as K562, than HHT used as reference.
The present invention provides cephalotaxanes having formula (I) 
wherein
W represents O or NH
Q represents an unbranched or branched, saturated or unsaturated or aromatic, cyclic or acyclic or heterocyclic hydrocarboned radical containing 1 to 30 carbon atoms including or not heteroatom(s),
R1 is H, OH, OMe, Oxe2x80x94(C1-C30)alkyl, O-aryl(C1-C30)alkyl, Oxe2x80x94(C2-C30)alkenyl, Oxe2x80x94(C3-C30)cycloalkyl or null and R2 is H or OH, or R1, R2 form together xe2x80x94Oxe2x80x94,
R3xe2x95x90R4=OMe or R3 and R4 form together xe2x80x94OCH2Oxe2x80x94,
R is H, C1-C30alkyl or O-protecting group and R6 represents an unbranched or branched, saturated or unsaturated or aromatic, cyclic or acyclic or heterocyclic hydrocarboned radical containing 1 to 30 carbon atoms including or not heteroatom(s), or R and R6 form together xe2x80x94CMe2xe2x80x94,
n is 0 to 8,
R5 is H, OH, OMe, Oxe2x80x94(C1-C30)alkyl, O-aryl(C1-C30)alkyl, Oxe2x80x94(C2-C30)alkenyl, Oxe2x80x94(C3-C30)cycloalkyl or O-aryl,
the doted line is null or forms a double bond depending on the meaning of R1, except for compounds where
W represents O, the doted line forms a double bond, R1 is null, R2 is H, R3 and R4 represent xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94, R5 is OMe, Q=CO2R7 and
1.) R=H, R6=xe2x80x94(Cxe2x80x94OH)Me2, n=2 or 3, R7=Me or H,
2.) R=H, R6=xe2x80x94(Cxe2x80x94H)Me2, n=2 to 4, R7=Me,
3.) R=H, R6=xe2x80x94(Cxe2x80x94H)Me2, n=1 or 2, R7=H,
4.) R=H, R6=Ph, n=1 to 3, R7=Me,
5.) R=H, R6=xe2x80x94CHxe2x95x90CHxe2x80x94Ph, n=0, R7=Me,
6.) R=H, R6=CH3, n=4, R7=Me,
7.) R and R6 form together xe2x80x94CMe2xe2x80x94, n=2 or 3, R7=Me,
W represents O, the doted line forms a double bond, R1 is null, R2 is OH, R3 and R4 represent xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94, R5 is OMe and R=H, R6=xe2x80x94(Cxe2x80x94H)Me2, n=2 or 3, R7=Me
W represents O, the doted line is null, R1 and R2 represent xe2x80x94Oxe2x80x94, R3 and R4 represent xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94, R5 is OMe and R=H, R6=xe2x80x94(Cxe2x80x94H)Me2, n=2, R7=Me.
The term xe2x80x9cO-Protecting groupxe2x80x9d as used in the present invention refers to a substituent which protects hydroxyl groups against undesirable reactions during synthetic procedures such as those O-protecting groups disclosed in Greene, xe2x80x9cProtective Groups In Organic synthesisxe2x80x9d, (John Wiley and Sons, New York (1981)). O-protecting groups comprise substituted methyl ethers, for example, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl, 2-(trimethylsilyl) ethoxymethyl, t-butyl, benzyl and triphenylmethyl, tetrahydropyranyl ethers, substituted ethyl ethers, for example, 2,2,2-trichloroethyl, silyl ethers, for example, trimethylsilyl, t-butyidimethylsilyl and t-butyldiphenylsilyl; and esters prepared by reacting the hydroxyl group with a carboxylic acid for example, acetate, propionate, benzoate and the like.
The term xe2x80x9cC1-C30alkylxe2x80x9d as used in the present invention refers to straight or branched chain substituted or unsubstituted alkyl radicals containing from 1 to 30 carbon atoms including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl and the like.
The term xe2x80x9cC2-C30alkenylxe2x80x9d as used in the present invention refers to straight or branched chain substituted or unsubstituted alkenyl radicals containing from 1 to 30 carbon atoms including, but not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like.
The term xe2x80x9carylxe2x80x9d as used in the present invention refers to a monocyclic or bicyclic carbocyclic ring system having one or more aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and the like. Aryl groups can be unsubstituted or substituted with one or more substituents.
The term xe2x80x9caryl(C1-C30)alkylxe2x80x9d as used in the present invention refers to an aryl group such as defined above appended to a C1-C30alkyl radical such as defined above, for example, benzyl and the like.
The term xe2x80x9cC3-C30cycloalkylxe2x80x9d as used herein refers to a carbocyclic ring having 3 to 30 carbon atoms including, but not limited to, cyclopropyl, cyclopentyl, cyclohexyl and the like. Cycloalkyl groups can be unsubstituted or substituted with one or more substituents.
An advantageous embodiment provides compounds of formula (I) wherein
Q=COZxe2x80x94R8,
Z=O, S, or NH, and 
or Zxe2x80x94R8 is NR12R13, R12 and R13 representing respectively R9 and R10,
R9, R10, R11 are independently H, C1-C30alkyl, C3-C30cycloalkyl, aryl, aryl-(C1-C30)-alkyl, C2-C30alkenyl, C2-C30alkynyl, C1-C30trihalogenoalkyl, C1-C30alkylamino-(C1-C30)-alkyl, C1-C30dialkylamino-(C1-C30)-alkyl, or amino-(C1-C30)-alkyl, or 
xe2x80x83where R14, R15, R16 are independently H, halogen, C1-C30 alkyl, C3-C30 cycloalkyl, aryl, aryl-(C1-C30)-alkyl, C2-C30 alkenyl or C2-C30 alkynyl, C1-C30 trihalogenoalkyl, m is 0 to 4,
each of these groups including or not heteroatom(s), such as in particular S, N or O.
The term xe2x80x9cC2-C30alkynylxe2x80x9d as used in the present invention refers to straight or branched chain substituted or unsubstituted alkynyl radicals containing from 1 to 30 carbon atoms including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl and the like.
The term xe2x80x9cC1-C30trihalogenoalkylxe2x80x9d as used in the present invention refers to straight or branched chain alkyl radicals containing from 1 to 30 carbon atoms substituted by three halogen atoms.
The term xe2x80x9cC1-C30alkylaminoxe2x80x9d as used in the present invention refers to R18NHxe2x80x94 wherein R18 is a C1-C30alkyl group such as defined above.
The term xe2x80x9cC1-C30dialkylaminoxe2x80x9d as used herein refers to R19R20Nxe2x80x94 wherein R19 and R20 are independently selected from C1-C30alkyl such as defined above.
The term xe2x80x9cC1-C30aminoalkylxe2x80x9d as used herein refers to a C1-C30alkyl radical such as defined above to which is appended an amino group (xe2x80x94NH2).
The term xe2x80x9cC1-C30alkylamino-(C1-C30)-alkylxe2x80x9d as used herein refers to a C1-C30alkyl radical such as defined above to which is appended an C1-C30alkylamino group such as defined above.
The term xe2x80x9cC1-C30dialkylamino-(C1-C30)-alkylxe2x80x9d as used herein refers to a C1-C30alkyl radical such as defined above to which is appended an C1-C30dialkylamino group such as defined above.
Another advantageous embodiment provides compounds of formula (I) wherein:
R6=xe2x80x94(Cxe2x80x94Y)Me2, xe2x80x94CHxe2x95x90CMe2, or an aryl group or R and R6 form together xe2x80x94CMe2xe2x80x94, Y=H, OH or halogen.
A further advantageous embodiment provides compounds of formula (I) wherein.
the doted line forms a double bond
R1 is null
R2 is H
R3 and R4 represent xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94
R5 is OMe.
A further aspect of the invention provides compounds of formula (I) wherein:
the doted line is null
R1 and R2 represent xe2x80x94Oxe2x80x94
R3 and R4 represent xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94
R5 is OMe.
Yet, a further preferred embodiment provides compounds of formula (I) wherein n=1 to 3.
Another further aspect of the invention provides compounds of formula (I) wherein W represents O.
Advantageously, a compound according to the present invention is selected from the group consisting of the following compounds 1 to 65: 
Th advantageous compounds of the present invention are the one of formula (I) wherein Q represents COZR8, Z is such as described above and R8 has 6 carbon atoms without any hydrophilic group.
Some of the compounds described herein contain one or more centers of asymmetry and may thus give rise to diastereoisomers and optical isomers. The present invention is meant to comprehend such possible isomers or diastereoisomers alone or in a mixture, such as for example their racemic mixture.
Some of the compounds described herein contain olefinic double bonds and are meant to include both E and Z geometric isomers.
Another aspect of the invention is a process for preparing a compound of formula (I).
A. In the case where W represents NH, it comprises the following steps A1 to A2:
A1: reacting cephalotaxines of formula V: 
xe2x80x83wherein
R1 is H, OH, OMe, Oxe2x80x94(C1-C30)alkyl, O-aryl(C1-C30)alkyl, Oxe2x80x94(C2-C30)alkenyl, Oxe2x80x94(C3-C30)cycloalkyl or null and R2 is H or OH, or R1, R2 form together xe2x80x94Oxe2x80x94,
R3xe2x95x90R4=OMe or R3 and R4 form together xe2x80x94OCH2Oxe2x80x94,
R5 is H, OH, OMe, Oxe2x80x94(C1-C30)alkyl, O-aryl(C1-C30)alkyl, Oxe2x80x94(C2-C30)alkenyl, Oxe2x80x94(C3-C30)cycloalkyl or O-aryl,
the doted line is null or forms a double bond depending on the meaning of R1 
xe2x80x83by methods known by the one skilled in the art to obtain the compounds of formula VI 
xe2x80x83wherein
R1 is H, OH, OMe, Oxe2x80x94(C1-C30)alkyl, O-aryl(C1-C30)alkyl, Oxe2x80x94(C2-C30)alkenyl, Oxe2x80x94(C3-C30)cycloalkyl or null and R2 is H or OH, or R1, R2 form together xe2x80x94Oxe2x80x94,
R3xe2x95x90R4=OMe or R3 and R4 form together xe2x80x94OCH2Oxe2x80x94,
R5 is H, OH, OMe, Oxe2x80x94(C1-C30)alkyl, O-aryl(C1-C30)alkyl, Oxe2x80x94(C2-C30)alkenyl, Oxe2x80x94(C3-C30)cycloalkyl or O-aryl,
the doted line is null or forms a double bond depending on the meaning of R1 
xe2x80x83For example, such methods are the following:
substituting the hydroxyl group of the cephalotaxines of formula V by an halogen or a tosylate by methods known by the one skilled in the art (such as by using SOCl2), then substituting the halogen or the tosylate by the group NH2 by methods known by the one skilled in the art (such as 1) the use of gaseous ammoniac in methylene chloride at 0xc2x0C. and 1 atm or 2) the use of NaN3 in DMF followed by 21) a catalytic hydogenolysis with for example Pd/C as the catalyst or 22) the use of LiAlH4) to obtain the compounds of formula VI.
A2: reacting the compounds of formula VI by the methods disclosed in WO 99/48894.
B. In the case where W represents O, Q=COR8 and R8 is such as described above, it comprises the step of reacting harringtonines or homoharringtonines commercially available (by SIGMA) with organometallic compounds, advantageously organolithium or grignard reagent.
C. In the case where W represents O, Q=CH2ZR8 or CH2R8 and Z and R8 are such as described above, it comprises the step of reducing harringtonines or homoharringtonines commercially available (by SIGMA) with an hydride, advantageously boron or aluminium hydride
D. In the case where W represents O, Q=COZxe2x80x94R8 and Z and R8 are such as described above, it comprises the following steps i) then ii),
i) hydrolyzing selectively the compound of formula (IV) which is available commercially 
xe2x80x83wherein
R1 is H, OH, OMe, Oxe2x80x94(C1-C30)-alkyl, O-aryl-(C1-C30)-alkyl, Oxe2x80x94(C2-C30)-alkenyl, Oxe2x80x94(C3-C30)-cycloalkyl or null and R2 is H or OH, or R1 and R2 form together xe2x80x94Oxe2x80x94
R3xe2x95x90R4=OMe or R3 and R4 form together xe2x80x94OCH2Oxe2x80x94
R is H, C1-C30alkyl or O-protecting group and R6 represents an unbranched or branched, saturated or unsaturated or aromatic, cyclic or acyclic or heterocyclic hydrocarboned radical containing 1 to 30 carbon atoms, including or not heteroatom(s), or R and R6 form together xe2x80x94CMe2xe2x80x94
n is 0 to 8.
R5 is H, OH, OMe, Oxe2x80x94(C1-C30)-alkyl, O-aryl-(C1-C30)-alkyl, Oxe2x80x94(C2-C30)-alkenyl, Oxe2x80x94(C3-C30)-cycloalkyl or O-aryl.
the doted line is null or forms a double bond depending on the meaning of R1, with an agent such as mineral hydroxide, advantageously lithium, potassium or sodium hydroxide, in hydro-organic solvent mixture to give as reaction product, amphoteric acid of formula (III) 
xe2x80x83wherein R1 to R5, R and R6 are defined as above,
ii) performing the esterification of the above obtained amphoteric acid of formula (III) with an esterification agent and a compound of formula R8xe2x80x94ZH, R8 and Z being defined as above
xe2x80x83and wherein the steps i) and ii) are carried out successively or simultaneously.
Advantageously the compounds of the following formula (II): 
wherein
R1 is H, OH, OMe, Oxe2x80x94(C1-C30)-alkyl, O-aryl-(C1-C30)-alkyl, Oxe2x80x94(C2-C30)-alkenyl, Oxe2x80x94(C3-C30)-cycloalkyl or null and R2 is H or OH, or R1 and R2 form together xe2x80x94Oxe2x80x94
R3xe2x95x90R4=OMe or R3 and R4 form together xe2x80x94OCH2Oxe2x80x94
R is H, C1-C30alkyl or O-protecting group and R6 represents an unbranched or branched, saturated or unsaturated or aromatic, cyclic or acyclic or heterocyclic hydrocarboned radical containing 1 to 30 carbon atoms, including or not heteroatom(s), or R and R6 form together xe2x80x94CMe2xe2x80x94
n is 0 to 8.
R5 is H, OH, OMe, Oxe2x80x94(C1-C30)-alkyl, O-aryl-(C1-C30)-alkyl, Oxe2x80x94(C2-C30)-alkenyl, Oxe2x80x94(C3-C30)-cycloalkyl or O-aryl.
the doted line is null or forms a double bond depending on the meaning of R1,
Z=O or S, and
R17 represents C1-C30alkyl, C2-C30alkenyl, C3-C30cycloalkyl, C2-C30alkynyl, aryl-(C1-C30)-alkyl or aryl and advantageously methyl or ethyl, can also be used instead of the compounds of formula IV to prepare, according to the process A, the product of formula (I).
Advantageously, the esterification agent in process A is a lewis acid or a protonic acid.
In an advantageous embodiment of the process A according to the present invention, the amphoteric acid of formula (III) is activated with an imide or by formation of a mixte anhydride or an acid chloride.
Advantageously, the imide is dicyclohexylcarbodiimide or diisopropylcarbodiimide
More advantageously the mixte anhydride is formed with 2,4,6-trichlorobenzoic acid by contact with 2,4,6-trichlorobenzoyl chloride in the presence of a base.
In another advantageous embodiment of the process A according to the present invention, the steps i) and ii) are carried out simultaneously, without isolation of the amphoteric acid of formula (III), via a reaction of transesterification performed in presence of an acidic or basic catalyst.
Advantageously, the catalyst is a base, such as an alkaline hydride.
More advantageously, the catalyst is a lewis acid or a protonic acid.
Given their pharmacological properties, the compounds of the present invention may be used in human therapy in the treatment of cancer pathology.
This invention includes also a pharmaceutical composition which comprises a therapeutically effective amount of at least one compound according to the present invention with one or more pharmaceutically acceptable carriers, excipients or diluents.
These pharmaceutical compositions may be formulated for oral, intravenous or subcutaneous administration.
Another aspect of the invention is the use of at least one compound according to the present invention or of the pharmaceutical composition as described above as (i) chemotherapeutic agent, (ii) enhancer of other chemotherapeutic agents (iii) for inhibiting tumors growth, (iv) for inhibiting mammalian parasites,
Yet, another aspect of the invention is the use of at least one compound according to the present invention for the preparation of a pharmaceutical composition as (i) chemotherapeutic agent, (ii) enhancer of other chemotherapeutic agents (iii) for inhibiting tumors growth, (iv) for inhibiting mammalian parasites, or (v) as reversal agents in particular of harringtonines. (a reversal agent is an agent able to reverse the cell multiresistance phenomenon).
Finally, the present invention describes a method for treating mammalian tumors which comprises administering to a mammal a therapeutically effective amount of at least one compound according to the present invention.
Another advantages of the compounds according to the present invention, is its activity on leukemic cell lines exhibiting resistance to other agents including harringtonines.